Polyketides natural products have exhibited vast pharmacological properties resulting in a $15 billion dollars industry 1,2. The biosynthetic mechanism of these versatile compounds is not well understood. By exploring and understanding the mechanisms that lead to polyketide biosynthesis, we hope to eventually bioengineer new polyketide therapeutics through in vitro recombination of polyketide synthase (PKS) to efficiently synthesize large quantities of the desired polyketide drug. Our long-term goal is to bioengineer polyketide enzymes to create new therapeutics. There are at least three types of PKSs, but the focus of this proposal is type II PKSs, which consist of five to ten enzymes that conduct chain elongations and modifications iteratively. The overall objective of this application is to provide structural insight into the aromatase/cyclase (ARO/CYC) of type II PKSs. Our central hypothesis is that we can apply structure-based mutagenesis to change the product outcome. Thus our purposed research is relevant to the NIH's mission that pertains to an innovative approach for ultimately protecting and improving health. Guided by our preliminary data, the hypothesis will be tested by two specific aims: 1) Determining the crystal structures of the polyketide di-domain aromatase/cyclase (ARO/CYC); and 2) Engineering the di-domain ARO/CYC to rationally control first-ring cyclization specificity. The proposed studies are significant, because there is a need to determine the molecular basis of cyclization specificities by di-domain ARO/CYCs in type II PKSs. This knowledge gap has so far prevented the development of new aromatic polyketides with new cyclization patterns.